Libraries of peptide conjugates and methods for making them

Abstract

The invention relates to peptide conjugates including at least one turn inducer wherein the turn inducer comprises a 5-7 membered saturated or unsaturated nitrogen containing heterocyclic ring and methods of making the peptides. Libraries of these peptides, methods of making the libraries are also described and methods of screening the libraries for therapeutic activity are also described.

1 . A library of peptide conjugates comprising two or more different peptide conjugates represented by formula (I): 
       
         
           
           
               
               
           
         
         wherein: 
         A and any B present are independently selected from a 5-7 membered saturated or unsaturated nitrogen-containing heterocyclic ring; 
         R 1  is an amino acid or a peptide having 2 to 5 amino acid residues, wherein the amino acid or peptide is optionally capped with a C-terminal capping group; 
         one of R 2  and R 3  is an amino acid or a peptide having 2 to 5 amino acid residues wherein the amino acid or peptide is optionally capped with an N-terminal capping group; 
         the other of R 2  and R 3  is hydrogen, —C 1-10 alkyl, —C 2-10 alkenyl, —C 2-10 alkynyl, —C 3-8  cycloalkyl, —C 0-6 alkylaryl, —C 0-6 alkylheterocyclyl, —C 0-6 alkylheteroaryl, —C 1-6 alkylCON(R a ) 2 , —C 1-6 alkylN(R a ) 2 , —C 1-6 alkylCO 2 R a , —C 1-6 alkylOR a , —C 1-6 alkylSR a , —C 1-6 alkylNR a C(═NR a )N(R a ) 2 , —C 1-6 alkylNR a SO 2 R a , —C 1-6 alkylSO 2 R a , —C 1-6 alkylOPO 3 R a , an acyl group (—COR a ) or a sulfonyl group (—SO 2 —R a ); wherein each R a  is independently selected from hydrogen, —C 1-6 alkyl, cycloalkyl, aryl, heterocyclyl and heteroaryl and wherein each alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl and heteroaryl group is optionally substituted with one or more of —C 1-6 alkyl, —C 2-6 alkenyl, —C 2-6 alkynyl, halo, —OH, —OC 1-6  alkyl, —NH 2 , —NH(C 1-6  alkyl), —N(C 1-6 alkyl) 2 , —NHC(═NH)NH 2 , oxo (═O), —CO 2 H, —CO 2 C 1-6 alkyl, —SH or —SC 1-6 alkyl; 
         each Q 1  is independently NH or absent; 
         when Q 1  is NH, Q 2  is C or CH, Q 3  is N and Q 4  is R 4 ; 
         when Q 1  is absent, Q 2  is N, Q 3  is C or CH and Q 4  is NHR 4 ; 
         each R 4  is independently selected from hydrogen, —C 1-10 alkyl, —C 2-10 alkenyl, —C 2-10 alkynyl, —C 3-8 cycloalkyl, —C 0-6 alkylaryl, —C 0-6 alkylheterocyclyl, —C 0-6 alkylheteroaryl, —C 1-6 alkylCON(R a ) 2 , —C 1-6 alkylN(R a ) 2 , —C 1-6 alkylCO 2 R a , —C 1-6 alkylOR a , —C 1-6 alkylSR a , —C 1-6 alkylNR a C(═NR a )N(R a ) 2 , —C 1-6 alkylNR a SO 2 R a , —C 1-6 alkylSO 2 R a , —C 1-6 alkylOPO 3 R a , an acyl group or a sulfonyl group; 
       
       wherein each R a  is independently selected from hydrogen, —C 1-6 alkyl, cycloalkyl, aryl, heterocyclyl and heteroaryl and wherein each alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl and heteroaryl group is optionally substituted with one or more of —C 1-6 alkyl, —C 2-6 alkenyl, —C 2-6 alkynyl, halo, —OH, —OC 1-6 alkyl, —NH 2 , —NH(C 1-6 alkyl), —N(C 1-6 alkyl) 2 , —NHC(═NH)NH 2 , oxo (═O), —CO 2 H, —CO 2 C 1-6 alkyl, —SH or —SC 1-6 alkyl;
 n is 0, 1 or 2; and 
 each p is independently 0 or 1; 
 wherein the carbonyl containing substituents (—(CH 2 ) p COQ 1 ) and/or (—(CH 2 ) p COR 1 ) are in an α-, β- or γ-position of the A and/or B rings with respect to the A and/or B ring nitrogen atoms; 
 or a salt thereof. 
 
     
     
         2 . A library of peptide conjugates according to  claim 1  wherein at least a portion of the peptide conjugates are cyclic as a result of cyclization between a side chain functional group in R 2  or R 3  or the N-terminus or the N-terminal capping group and a side chain functional group in R 1  or the C-terminus or C-terminal capping group. 
     
     
         3 . A library of peptide conjugates according to  claim 2  wherein the cyclic peptide conjugates comprise a disulfide, diseleno or sulfoselenium bond between a cysteine, homocysteine, penicillamine or selenocysteine residue in R 1  and a cysteine, homocysteine, penicillamine or selenocysteine residue or a thiol group in the N-terminal capping group of R 2  or R 3 . 
     
     
         4 . A library of peptide conjugates according to  claim 3  wherein the cyclic peptide conjugates comprise a disulfide bond or methylenedithio linker between a cysteine residue in R 1  and a cysteine residue in R 2  or R 3 . 
     
     
         5 . A library of peptide conjugates according to  claim 1  wherein ring A is a 5- or 6-membered saturated or unsaturated nitrogen-containing heterocyclic ring. 
     
     
         6 . A library of peptide conjugates according to  claim 5  wherein ring A is a 5- or 6-membered saturated nitrogen-containing heterocyclic ring. 
     
     
         7 . A library of peptide conjugates according to  claim 6  wherein ring A is pyrrolidine or piperidine. 
     
     
         8 . A library of peptide conjugates according to  claim 1  wherein n is 0. 
     
     
         9 . A library of peptide conjugates according to  claim 1  wherein R 1  is an amino acid residue or a peptide having 2 to 3 amino acid residues optionally capped with an amide. 
     
     
         10 . A library of peptide conjugates according to  claim 1  wherein at least a portion of the peptide conjugates have R 2  as an amino acid residue or a peptide having 2 to 3 amino acid residues and R 3  as hydrogen or an acyl group. 
     
     
         11 . A library of peptide conjugates according to  claim 1  wherein at least a portion of the peptide conjugates have R 3  as an amino acid residue or a peptide having 2 to 3 amino acid residues and R 2  as hydrogen or an acyl group. 
     
     
         12 . A library of peptide conjugates according to  claim 1  wherein at least a portion of the peptide conjugates have —C(O)R 1  in the α-position with respect to the ring nitrogen atom. 
     
     
         13 . A library of peptide conjugates according to  claim 1  wherein at least a portion of the peptide conjugates have an A ring which is a 6-membered ring and —C(O)R 1  is in γ-position with respect to the ring nitrogen. 
     
     
         14 . A library of peptide conjugates according to  claim 1  wherein at least a portion of the peptide conjugates have an acyl group R 2  or R 3  selected from —(C═O)R where R is —C 1-6 alkyl, —C 0-6 alkylcycloalkyl, —C 0-6 alkylNHC(═NH)NH 2 , —C 1-6 alkylCONH 2 , —C 1-6 alkylCO 2 H, —C 0-6 alkylSH, —C 1-6 alkylCO 2 H, —C 0-6 alkyl(heterocyclyl), —C 0-6 alkyl(heteroaryl), —C 0-6 alkylNH 2 , —C 0-6 alkylSC 1-6 alkyl, —C 0-6 alkylaryl and —C 0-6 alkylOH, wherein each aryl, heterocyclyl or heteroaryl group may be optionally substituted with —C 1-3 alkyl, —OH, —NH 2  or -oxo (C═O). 
     
     
         15 . A library of peptide conjugates according to  claim 1  comprising two or more peptide conjugates represented by formula III and/or formula IV 
       
         
           
           
               
               
           
         
         wherein 
         R 2a  and R 3a  are hydrogen, acyl, sulfonyl or —C 1-6 alkyl; 
         each R 10  is independently selected from an amino acid side chain; 
         R 11  is absent or is NR 13 H where R 13  is hydrogen, an N-terminal capping group or an amino acid residue or peptide having 2 or 3 amino acid residues optionally capped with an N-terminal capping group; 
         R 12  is absent or is C(O)R 14  where R 14  is —OH or —NH 2 ; and 
         L is a linker that forms a cyclic peptide; or a salt thereof. 
       
     
     
         16 . A library of peptide conjugates according to  claim 15  wherein L is a linker selected from —S—S—, —S—(CH 2 ) 1-3 —S—, —Se—Se—, —Se—S—, —S—Se—, —C(O)NH—, —NHC(O)—, —OC(O)—, —C(O)O—, —O—, —NH—, —S— or —CH═CH—. 
     
     
         17 . A library of peptide conjugates according to  claim 16  wherein L is —S—S—, —S—(CH 2 )—S—, —Se—Se—, —Se—S— or —S—Se—. 
     
     
         18 . A library of peptide conjugates according to  claim 15  wherein the peptides are represented by formula III and/or formula IV. 
     
     
         19 . A method of producing a focussed peptide conjugate library, said method comprising the steps of:
 i) identifying a bioactive turn-containing peptide and its target receptor or enzyme;   ii) identifying amino acid residues around the turn in the bioactive peptide;   iii) preparing a focussed library comprising two or more peptide conjugates of formula (V)   
       
         
           
           
               
               
           
         
         wherein A and any B present are independently selected from a 5-7 membered saturated or unsaturated nitrogen-containing heterocyclic ring; 
         R 1b  is an amino acid residue or a peptide of 2 to 5 residues wherein the amino acid residue or peptide is optionally capped with a C-terminal capping group; 
         one of R 2b  and R 3b  is hydrogen, a substituent selected from —C 1-10 alkyl, —C 2-10 alkenyl, —C 2-10 alkynyl, —C 3-8 cycloalkyl, —C 0-6 alkylaryl, —C 0-6 alkylheterocyclyl, —C 0-6 alkylheteroaryl, —C 1-6 alkylCON(R a ) 2 , —C 1-6 alkylN(R a ) 2 , —C 1-6 alkylCO 2 R a , —C 1-6 alkylOR a , —C 1-6 alkylSR a , —C 1-6 alkylNR a C(═NR a )N(R a ) 2 , —C 1-6 alkylNR a SO 2 R a , —C 1-6 alkylSO 2 R a , —C 1-6 alkylOPO 3 R a , an acyl group or a sulfonyl group; wherein each R a  is independently selected from hydrogen, —C 1-6 alkyl, cycloalkyl, aryl, heterocyclyl and heteroaryl and wherein each alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl and heteroaryl group is optionally substituted with one or more of —C 1-6 alkyl, —C 2-6 alkenyl, —C 2-6 alkynyl, halo, —OH, —OC 1-6 alkyl, —NH 2 , —NH(C 1-6 alkyl), —N(C 1-6 alkyl) 2 , —NHC(═NH)NH 2 , oxo (═O), —CO 2 H, —CO 2 C 1-6 alkyl, —SH or —SC 1-6 alkyl; 
         the other of R 2b  and R 3b  is an amino acid or a peptide of 2 to 5 residues wherein the amino acid or peptide is optionally capped with an N-terminal capping group; 
         each Q 1b  is independently NH or absent; 
         when Q 1b  is NH, Q 2b  is C or CH, Q 3b  is N and Q 4b  is R 4b ; 
         when Q 1b  is absent, Q 2b  is N, Q 3b  is C or CH and Q 4b  is NHR 4b ; 
         each R 4b  is independently selected from hydrogen, a substituent selected from —C 1-10 alkyl, —C 2-10 alkenyl, —C 2-10 alkynyl, —C 3-8 cycloalkyl, —C 0-6 alkylaryl, —C 0-6 alkylheterocyclyl, —C 0-6 alkylheteroaryl, —C 1-6 alkylCON(R a ) 2 , —C 1-6 alkylN(R a ) 2 , —C 1-6 alkylCO 2 R a , —C 1-6 alkylOR a , —C 1-6 alkylSR a , —C 1-6 alkylNR a C(═NR a )N(R a ) 2 , —C 1-6 alkylNR a SO 2 R a , —C 1-6 alkylSO 2 R a , —C 1-6 alkylOPO 3 R a , an acyl group or a sulfonyl group; wherein each R a  is independently selected from hydrogen, —C 1-6 alkyl, cycloalkyl, aryl, heterocyclyl and heteroaryl and wherein each alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl and heteroaryl group is optionally substituted with one or more of —C 1-6 alkyl, —C 2-6 alkenyl, —C 2-6 alkynyl, halo, —OH, —OC 1-6 alkyl, —NH 2 , —NH(C 1-6 alkyl), —N(C 1-6 alkyl) 2 , —NHC(═NH)NH 2 , oxo (═O), —CO 2 H, —CO 2 C 1-6 alkyl, —SH or —SC 1-6 alkyl; 
         n is 0, 1 or 2; 
         each p is independently 0 or 1; 
         wherein at least one amino acid of R 1b , R 2b  or R 3b  is an amino acid that forms part of the peptide turn in the bioactive turn-containing peptide or an amino acid that is a conservative substitution thereof and/or at least one of R 2b , R 3b  or R 4b  is a substituent, acyl group or sulfonyl group that mimics the side chain of an amino acid residue that forms part of the peptide turn in the bioactive turn-containing peptide or a conservative substitution thereof; and 
         wherein the carbonyl containing substituents (—(CH 2 ) p COQ 1b ) and/or (—(CH 2 ) p COR 1b ) are in an α-, β- or γ-position of the A and/or B rings with respect to the A and/or B ring nitrogen atoms; or a salt thereof. 
       
     
     
         20 . A method of preparing a library of peptide conjugates comprising the steps of:
 i) preparing a first peptide attached to a compartmentalized solid phase support through a safety catch linker,   ii) introducing a turn inducer represented by the formula (II)   
       
         
           
           
               
               
           
         
         
           wherein A is a 5-7 membered saturated or unsaturated nitrogen-containing heterocyclic ring, p is 0 or 1, R 5  and R 6  are independently orthogonal amino protecting groups wherein at least one protecting group is stable under conditions used to deprotect the other amino protecting group, wherein the carboxylic acid or acetyl substituent is in the α-, β- or γ-position of the ring with respect to the ring nitrogen atom; 
         
         iii) deprotecting one of the amino protecting groups R 5  or R 6  on the N-terminal turn inducer; 
         iv) optionally repeating steps ii) and iii) one or two more times; 
         v) introducing a second peptide at the free amino group of the N-terminal turn inducer; 
         vi) deprotecting the remaining turn inducer protecting group(s), R 5  or one to three R 6 s, the N-terminal protecting group and side chain protecting groups; and 
         vii) cleaving the peptide conjugates from the compartmentalized solid support and linker;
 wherein the first peptide and second peptide independently comprise 1 to 5 amino acid residues; and 
 wherein at least one of preparing the first peptide, introducing the turn inducer(s), and introducing the amino acids of the second peptide involves a split and mix strategy to introduce variation into the amino acid sequence or turn inducer of the peptide conjugate. 
 
       
     
     
         21 . A method of preparing a library of peptide conjugates according to  claim 20  further comprising the step of cyclizing the peptide conjugate. 
     
     
         22 . A method of preparing a library of peptide conjugates according to  claim 21  wherein cyclization occurs between a cysteine, homocysteine, penicillamine or selenocysteine residue in the first peptide and a cysteine, homocysteine, penicillamine or selenocysteine residue or a thiol group in the N-terminal capping group in the second peptide. 
     
     
         23 . A method of preparing a library of peptide conjugates according to  claim 21  wherein cyclization occurs between a cysteine residue in the first peptide and a cysteine residue in the second peptide. 
     
     
         24 . A method of preparing a library of peptide conjugates according to  claim 23  wherein cyclization occurs after peptide conjugate cleavage from the solid phase support and linker. 
     
     
         25 . A method of preparing a library of peptide conjugates according to  claim 20  wherein the compartmentalized solid phase support is a resin coated Lantern. 
     
     
         26 . A method of preparing a library of peptide conjugates according to  claim 20  wherein the safety catch linker is SCAL linker. 
     
     
         27 . A method of preparing a library of peptide conjugates according to  claim 20  wherein the peptide conjugates are synthesized using BOC chemistry. 
     
     
         28 . A method of preparing a library of peptide conjugates according to  claim 20  wherein one of R 5  and R 6  is BOC and the other is Fmoc. 
     
     
         29 . A method of preparing a library of peptide conjugates according to  claim 20  further comprising acylating at least a portion of the free amino group of the turn inducer after deprotection in step vi) with an acylating agent. 
     
     
         30 . A method of preparing a library of peptide conjugates according to  claim 29 , wherein the acylating agent mimics the side chain of an amino acid. 
     
     
         31 . A method of preparing a library of peptide conjugates according to  claim 20  comprising the step of:
 i) preparing a first peptide attached to a lantern solid phase support through a SCAL linker; 
 ii) introducing a turn inducer represented by the formula (IIa) 
 
       
         
           
           
               
               
           
         
         
           wherein one of R 5  and R 6  is BOC and the other is Fmoc, p is 0 or 1; 
         
         iii) deprotecting the BOC group; 
         iv) introducing a second peptide at the free amino group of the turn inducer; 
         v) deprotecting the Fmoc group from the turn inducer to provide a free amino group; 
         vi) deprotecting the N-terminal protecting groups and the side chain protecting groups; and 
         vii) cleaving the peptide conjugates from the lantern and linker;
 wherein the first peptide and the second peptide independently comprise the two amino acid residues in which the first amino acid residue introduced into the first peptide and the second amino acid residue introduced into the second peptide are residues have a thiol or selenol group, optionally protected; 
 the method further comprising cyclizing the peptide conjugate to form a disulfide, diseleno or sulfoseleno bond, and 
 wherein at least one of preparing the first peptide, introducing the turn inducer and introducing the second peptide involves a split and mix strategy to introduce variation into the amino acid sequence or turn inducer of the peptide conjugate. 
 
       
     
     
         32 . A method of preparing a library of peptide conjugates according to  claim 20  comprising the step of:
 (i) preparing a first peptide attached to a lantern solid phase support through a SCAL linker; 
 (ii) introducing a turn inducer represented by the formula (IIb) 
 
       
         
           
           
               
               
           
         
         wherein one of R 5  and R 6  is BOC and the other is Fmoc, p is 0 or 1; 
         (iii) deprotecting the BOC group; 
         (iv) introducing a second peptide at the free amino group of the turn inducer; 
         (v) deprotecting the Fmoc group from the turn inducer to provide a free amino group; 
         (vi) deprotecting the N-terminal protecting groups and the side chain protecting groups; and 
         (vii) cleaving the peptide conjugates from the lantern and linker; 
         wherein the first peptide and the second peptide independently comprise the two amino acid residues in which the first amino acid residue introduced into the first peptide and the second amino acid residue introduced into the second peptide are residues have a thiol or selenol group, optionally protected; 
         the method further comprising cyclizing the peptide conjugate to form a disulfide, diseleno or sulfoseleno bond, and 
         wherein at least one of preparing the first peptide, introducing the turn inducer and introducing the second peptide involves a split and mix strategy to introduce variation into the amino acid sequence or turn inducer of the peptide conjugate. 
       
     
     
         33 . A method according to  claim 31  further comprising acylating at least a portion of the free amino group of the turn inducer after deprotection in step v), with an acylating agent. 
     
     
         34 . A method according to  claim 33 , wherein the acylating agent mimics the side chain of an amino acid. 
     
     
         35 . (canceled) 
     
     
         36 . (canceled) 
     
     
         37 . (canceled) 
     
     
         38 . (canceled) 
     
     
         39 . A method according to  claim 32  further comprising acylating at least a portion of the free amino group of the turn inducer after deprotection in step v), with an acylating agent. 
     
     
         40 . A method according to  claim 39 , wherein the acylating agent mimics the side chain of an amino acid.